Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore.

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

Channel-independent function of UNC-9/Innexin in spatial arrangement of GABAergic synapses in C. elegans.

Precise synaptic connection of neurons with their targets is essential for the proper functioning of the nervous system. A plethora of signaling pathways act in concert to mediate the precise spatial arrangement of synaptic connections. Here we show a novel role for a gap junction protein in controlling tiled synaptic arrangement in the GABAergic motor neurons in Caenorhabditis elegans, in which their axons and synapses overlap minimally with their neighboring neurons within the same class. We found that while EGL-20/Wnt controls axonal tiling, their presynaptic tiling is mediated by a gap junction protein UNC-9/Innexin, that is localized at the presynaptic tiling border between neighboring dorsal D-type GABAergic motor neurons. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in controlling tiled presynaptic patterning. While gap junctions are crucial for the proper functioning of the nervous system as channels, our finding uncovered the novel channel-independent role of UNC-9 in synapse patterning.

Rapid image deconvolution and multiview fusion for optical microscopy.

The contrast and resolution of images obtained with optical microscopes can be improved by deconvolution and computational fusion of multiple views of the same sample, but these methods are computationally expensive for large datasets. Here we describe theoretical and practical advances in algorithm and software design that result in image processing times that are tenfold to several thousand fold faster than with previous methods. First, we show that an 'unmatched back projector' accelerates deconvolution relative to the classic Richardson-Lucy algorithm by at least tenfold. Second, three-dimensional image-based registration with a graphics processing unit enhances processing speed 10- to 100-fold over CPU processing. Third, deep learning can provide further acceleration, particularly for deconvolution with spatially varying point spread functions. We illustrate our methods from the subcellular to millimeter spatial scale on diverse samples, including single cells, embryos and cleared tissue. Finally, we show performance enhancement on recently developed microscopes that have improved spatial resolution, including dual-view cleared-tissue light-sheet microscopes and reflective lattice light-sheet microscopes.

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Semaphorin and Eph receptor signaling guide a series of cell movements for ventral enclosure in C. elegans.

BACKGROUND: In the last stage of the Caenorhabditis elegans body wall closure, an open pocket in the epidermis is closed by the migration of marginal epidermal P/pocket cells to the ventral midline. The cellular and molecular mechanisms of this closure remain unknown. RESULTS: Cells within the pocket align to form a bridge for migration of contralateral P cell pair P9/10 L,R (and neighboring P cells) to the midline. Bridge formation involves rearrangement of five sister pairs of PLX-2/plexin and VAB-1/Eph receptor expressing "plexin band" cells, of which three pairs form a scaffold for bridge assembly and two pairs form the bridge. Bridge formation requires VAB-1 kinase-dependent extension of presumptive bridge cells over scaffold cells toward the ventral midline. An unassembled vab-1 null mutant bridge obstructs P cell migration, which is largely overcome by plexin band expression of VAB-1 or VAB-1(delC) (a kinase deletion of VAB-1). VAB-1 also functions redundantly with MAB-20/semaphorin to prevent perdurant gaps between sister plexin band cells that block P cell migration. CONCLUSIONS: The Eph receptor mediates cellular extensions required for bridge formation, independently facilitates P cell migration to the midline, and functions redundantly with PLX-2/plexin to prevent gaps in the bridge used for P9/10 cell migration in body wall closure.

Integration of semaphorin-2A/MAB-20, ephrin-4, and UNC-129 TGF-beta signaling pathways regulates sorting of distinct sensory rays in C. elegans.

Semaphorins and ephrins are axon guidance cues. In C. elegans, semaphorin-2a/mab-20 and ephrin-4/efn-4/mab-26 also regulate cell sorting to form distinct rays in the male tail. Several erf (enhancer of ray fusion) mutations were identified in a mab-20 enhancer screen. Mutants of plexin-2 (plx-2) and unc-129, which encodes an axon guiding TGF-beta, were also found to be erfs. Genetic analyses show that plx-2 and mab-20 function in the same pathway, as expected if PLX-2 is a receptor for MAB-20. Surprisingly, MAB-20 also signals in a parallel pathway that requires efn-4. This signal utilizes a non-plexin receptor. The expression of plx-2, efn-4, and unc-129 in subsets of 3-cell sensory ray clusters likely mediates the ray-specific cell sorting functions of the ubiquitously expressed mab-20. We present a model for the integrated control of TGF-beta, semaphorin, and ephrin signaling in the sorting of cell clusters into distinct rays in the developing male tail.